毛細管電泳在DNA限制酵素圖譜製圖上的應用

Abstract

在從是基因或DNA的分析及圖譜製定實驗中,最大的瓶頸在於基因或DNA的 分離和偵測o傳統的膠電泳耗時甚久且操作手續繁雜,無法自動化o放射性 同位素偵測法為另一製定DNA圖譜常用的工具,然而其操作也頗費時,並且 有安全上的顧慮o毛細管電泳高效率的分離能力,以及自動化的優點,近幾 年來在分析技術方面迅速竄起,尤其應用在生化方面的研究更能展現出它 的特色o本論文探討利用限制酵素量及反應時間的調控,間接操作限制酵素 的活性,使得它對DNA分子,可以完全消化或部份消化成數個DNA片段,再藉 著末端耦合螢光染劑的方式,來做DNA圖譜的研究o這種方法除了增加偵測 的靈敏度之外,還具備迅速,簡單等多項優點o Separation and detection of DNA restriction fragments are the essential and major steps in gene analysis or physical mapping of genes. Slab gel electrophoresis is the trational way to separate DNA fragments,however, the method is time consuming ,labor intensive, hard to quantitate, and large sample quantity is required for analysis. To detect DNA fragment in a slab gel either ethidium bromide or radioisotope is emploted and these chemicals can pose futher health hazardous problem. Capillary electrophoresis is an automated separation technique offering the benefits of rapid and high resolution separation of DNA molecules. Capillary electrophoresis has been a maturenique for separating proteins, peptide, organic chemicals, and small fragments separations, several hurdles imposed by the nature of large DNA molecules needed to be overcome before the technique can be realized. In this thesis, I explore several capillary electrophoresis conditions to separate large DNA restriction fragments and apply the technique to DNA restriction mapping which is an essential tool to many molecular biology labs. Laser induced fluorescence detection and partial digest mapping method are adopted and modified to map DNA fragments by capillarytrophoresis. The pros and cons of capillary electrophoresis in DNA mapping as compared to the tradtional slab gel electrophoresis are discussed by using pBluescript SK(+) phagemid DNA as a model. A restriction map of human interleukine 5 receptor gene is constructed using capillary electrophoresis and the technique developed in this thesis to demonstrate and conclude the feasibility of this thesis project.